The long-term goal of this proposal is to transition to an independent research career by defining disease mechanisms in autoimmune inflammatory myopathies (AIM), with a view to improving the diagnosis and treatment of these disorders. These diseases, which include dermatomyositis (DM) and polymyositis (PM), are characterized by muscle weakness, muscle inflammation, and autoantibodies. The mechanisms underlying AIM are poorly understood, and available therapies have deleterious side-effects and do not induce permanent remission. Defining disease mechanisms may allow development of specific and effective therapies. In AIM, specific autoantibodies are strongly associated with distinct clinical phenotypes. The presence of anti-Mi-2 autoantibodies exclusively in patients with DM is a prominent example and understanding the basis of this association may shed light on underlying disease mechanisms. Recent data show low levels of myositis autoantigens (including Mi-2, a chromatin remodeling enzyme) in normal muscle. In contrast, myositis muscle has high myositis autoantigen levels, with enhanced expression particularly in regenerating cells. Interestingly, Mi-2 expression is only increased in DM and not PM muscle. Preliminary data from an in vitro myoblast differentiation system show that high Mi-2 levels in proliferating myoblasts are down-regulated during differentiation. Importantly, Mi-2 silencing with siRNA accelerated myoblast differentiation. Thus, we propose that Mi-2 may have a central role in myogenesis by inhibiting myoblast differentiation. In Aim 1, we will use this in vitro system to determine the effects of Mi-2 sjlencing and over- expression on myoblast proliferation and differentiation, muscle regulatory factor expression, and myotube formation. In Aim 2, we will use chromatin immunoprecipitation assays to identify candidate muscle-specific gene promoters that bind Mi-2. To broadly characterize the role of Mi-2 in regulating chromatin structure during myogenesis, we will perform large-scale ChlPSeq assays to define the entire Mi-2-DNA interactome in myoblasts and myotubes. Based on our understanding of Mi-2, its role in muscle differentiation, and its down-stream targets, we will undertake a detailed analysis of human muscle biopsy specimens in Aim 3. By defining normal and dysregulated muscle regeneration patterns in myositis and other muscle diseases, we hope to elucidate pathologic mechanisms and develop novel diagnostic tools.